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We read with interest the article by Passamonti et al,1 reporting the performance of two different faecal immunochemical tests (FITs) highlighting the importance of standardisation and validation of screening methodologies. Conventionally, laboratory-based FIT is the preferred approach in testing for occult blood in faeces, which includes colorectal cancer screening programmes.2–4 The potential of preserving stable faecal samples in a widely used FIT buffer for microbiome research would enable prospective microbiome studies in generally healthy subjects undergoing colorectal cancer screening.
For this purpose, we evaluated faecal sample stability in the commonly used OC-Sensor (Eiken Chemical, Tokyo, Japan) under various storage conditions. Faecal samples from five healthy adult individuals were used for the analysis and exposed to 16 different conditions: immediately frozen at −86°C (with FIT (wFIT)/without FIT (woFIT)); immediately frozen at −20°C (wFIT/woFIT); wFIT stored at 4°C for 1, 2, 7, 14 days; stored at 20°C for 1, 2, 7, 14 days; stored at 30°C for 1, 2, 7 days and lastly stored at 4°C for 2 days and at 20°C for additional 2 days. Shotgun metagenomic analysis was performed by Illumina 2500, while taxonomic compositions were determined by 16S rRNA analysis employing Illumina MiSeq and Ion Torrent PGM.
Our first attempt to extract DNA from wFIT samples failed due to low amount of DNA obtained. Thus, lyophilisation for all of the wFIT samples, except for woFIT samples, was applied, increasing the yield of DNA up to 30 times.
As shown by the figure 1, the Shannon index in both platforms displayed a clear pattern of decreased diversity during prolonged storage. While testing for differences between samples we discovered that woFIT samples, wFIT samples stored at 4°C for 1 and 2 days, wFIT samples stored at 20°C for 1 day from the Illumina MiSeq data significantly differ from wFIT samples stored at 30°C for 7 days (one-way analysis of variance; p<0.05).
The similarity matrix using unweighted principal coordinate analysis (PCoA, figure 2A,C) demonstrated that samples collected from each individual clustered together and was consistent in data gained from both sequencing platforms, suggesting that the biological effect outweighed the effect of the sample handling. Further, the weighted PCoA (figure 2B,D) displays that native samples tend to cluster closer while samples stored for prolonged periods tend to be located further away from the native ones.
Comparing observed operational taxonomic units (OTUs) between samples that were stored within various storage conditions, we were unable to identify any specific OTU cluster across all taxonomical levels that would significantly differ (Kruskal-Wallis test) between any storage condition. Nevertheless, we found that the relative abundances of Gram-negative bacteria tended to decrease while the abundances for most of the Gram-positive bacteria tended to increase over time. However, it is possible that even within native samples certain bacterial OTUs could fluctuate regardless of the sequencing platform employed.
Shotgun sequencing was performed on 12 samples (figure 2E) from four individuals: six woFIT samples (WO1–4) that were immediately stored at −86°C and six wFIT samples from three individuals (FIT1–4), stored for 2 days at 4°C and for 2 days at 20°C to imitate the typical sample treatment procedure when the material is being transported to a central laboratory by standard mail delivery. In addition to access the reproducibility of acquired data, technical replicates in the form of DNA that was extracted from independently collected samples from individuals 2 and 3 were included within this analysis. However, two wFIT samples (eg, FIT2.2 and FIT4) failed during the sequencing stage and therefore were excluded from further analysis. A similarity matrix was built with relative abundances to calculate the PCoA. Figure 2E illustrates the similarity between species for two types of sampling showing that results are not biased to the sampling procedures. Results indicate that sample similarities tended towards samples from the same subject rather than the storage method.
In this study, we sequenced microbiome samples employing two different sequencing platforms—Ion Torrent PGM that allows to analyse one variable region (V3) and to verify acquired results and also Illumina MiSeq platform that enables the analysis of two variable regions (V3–V4) at a time. Analysing absolute OTU frequencies within the samples, we did not find a significant difference between immediately frozen samples, which coincide with previous studies.5–8 Although previous study9 has found lower FIT stability measures, we observed some limitations as they extracted DNA from a small proportion of sample, while in our study design lyophilisation procedure was included allowing to extract DNA from whole volume. However, we did not include technical replicates for 16S rRNA analysis. Another critical aspect in FIT-oriented studies is the variety of available FIT test tubes in the market. Each FIT buffer from various manufacturers might contain slightly different ingredients and concentrations,10 which seems to be a trade secret and might alter the microbial composition. In our study, employed OC-Sensor is a reliable and convenient sampling device that can be used in large-scale microbiome studies on a population level even outside colorectal cancer screening programmes.
ML and LE contributed equally.
DG and SS contributed equally.
Contributors Study concept and design was created by ML and LE. Data collection was performed by DG, HW, IR-S, RH, IK, IE, GS. Bioinformatics analysis performed by DG, SS, IS, RP. DG and SS wrote the manuscript. Funding was obtained by LE, DF, JK and ML. All authors approved the final version for publication.
Funding The work was supported by the ERDF project ‘Long-term effects of H. pylori eradication on gastrointestinal tract microbiome and development of screening system for detection of extended-spectrum beta-lactamase coding genes within faeces samples’ (project no: 126.96.36.199/16/A/272) and in part by the Swedish Research Council (project no: K2013-56X-10848) and the Söderbergs Foundation.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Biomedical Ethics Committee of the Riga East University Hospital Support Foundation, approval no: 13-A/13.
Provenance and peer review Not commissioned; externally peer reviewed.
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